High-speed multi-color microscopy of repeating dynamic processes
Jungho Ohn, Jennifer Yang, Scott E. Fraser, Rusty Lansford, Michael Liebling
Genesis doi: 10.1002/dvg.20774
Images of multiply-labeled fluorescent samples provide unique insights into the localization of molecules, cells, and tissues. The ability to image multiple channels simultaneously at high speed without cross talk is limited to a few colors and requires dedicated multi-channel or multi-spectral detection procedures. Simpler microscopes, in which each color is imaged sequentially, produce a much lower frame rate. Here, we describe a technique to image, at high frame rate, multiply-labeled samples that have a repeating motion. We capture images in a single channel at a time over one full occurrence of the motion then repeat acquisition for other channels over subsequent occurrences. We finally build a high-speed multi-channel image sequence by combining the images after applying a normalized mutual information-based time registration procedure. We show that this technique is amenable to image the beating heart of a double-labeled embryonic quail in three channels (brightfield, yellow and mCherry fluorescent proteins) using a fluorescence wide-field microscope equipped with a single monochrome camera and without fast channel switching optics. We experimentally evaluate the accuracy of our method on image series from a two-channel confocal microscope.